Fig. 5. Redistribution of membrane-associated PI 3-kinase in heregulin-stimulated SKBR3 cells. (A) Cytosol and membrane fractions were prepared from treated and untreated SKBR3 cells, as described in the legend to Fig. 4. Samples were subjected to SDS-PAGE and immunoblotting with anti-p85 antibodies. (B) Immunoblotting evidence for the co-precipitation of p85 with ErbB3 (and very little co-precipation with EGFR or ErbB2). Treatment of cells is indicated. Membrane sheets in C-E were prepared from serum starved cells (C) or cells treated for 5 minutes with 3.2 nm heregulin (D,E); membranes were then double labeled for p85 (5-nm gold) and ErbB3 (10-nm gold). Bold arrows in C and D point to labeled ErbB3 label and fine arrows to labeled p85 label. Circles in E indicate multiple co-clusters of ErbB3 and p85. Bars, 0.1 µm.